Transdominant negative proto-oncogene

ABSTRACT

The present invention provides polynucleotide and polypeptide sequences for a trans-repressing protein of the Fos proto-oncogene family, where the polypeptide is characterized by having a leucine zipper domain and forming a heterodimer with a Jun related protein. This heterodimer is capable of biding to an AP-1 site and suppressing transcriptional transactivation of a promoter containing the AP-1 site.

This invention was made with Government support under NIH R35 CA 44360awarded by the National Institutes of Health. The Government has certainrights in the invention.

This is a continuation of application Ser. No. 07/710,862 filed on Jun.10, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of The Invention

This invention relates to unique proto-oncogene sequences derived usingrecombinant DNA technology and to the production and use of thesesequences as well as the expression products thereof.

2. Related Art

Advances in recombinant DNA technology have led to the discovery ofnormal cellular genes (proto-oncogenes) which control growth,development, and differentiation. Under certain circumstances,regulation of these genes is altered and they become oncogenes whichcause normal cells to assume neoplastic growth behavior. There areapproximately 40 known proto-oncogenes to date, which fall into variouscategories depending on their functional characteristics. These-include,(1) growth factors and growth factor receptors, (2) messengers ofintracellular signal transduction pathways, for example, between thecytoplasm and the nucleus, and (3) regulatory proteins influencing geneexpression and DNA replication.

Several oncogenes and proto-oncogenes are known to encode more than oneproduct by using alternatively spliced mRNAs. These include the SV40 Tantigen gene, c-src, c-Ha-ras, c-abl and c-myb. Alternative splicingincludes or excludes particular coding sequences within the mRNA, whichis eventually translated. Consequently, the biological function of asingle gene can be expanded by the splicing choice. Previous reportshave shown that an alternatively spliced c-myb mRNA encodes a truncatedform of the c-myb p75 which includes the DNA binding region and nuclearlocalization signal present in c-myb protein, but lacks regulatoryregions required for transcriptional activation (Weber, et al., Science,249:1291, 1990). The truncated protein has been shown to interfere withthe function of c-myb during differentiation of mouse erythroid leukemiacells.

Many proteins cooperate with each other in the activation oftranscription from specific promoters. Through this cooperation, thegene can be transcribed and a protein product generated. Members of theFos proto-oncogene family, along with members of the Jun gene family,form stable complexes which bind to DNA at a specific site designatedAP-1. The AP-1 site is located in the promoter region of a large numberof genes. Binding of the Fos/Jun complex activates transcription of agene associated with an AP-1 site. In cells that have lost their growthregulatory mechanisms, it can be envisioned that this Fos/Jun complexmay "sit" on the AP-1 site, causing over expression of a particulargene. Since many cell proliferative disorders result from the overexpression of an otherwise normal gene, such as a proto-oncogene, itwould be desirable to identify methods which interfere with theexcessive activation of these genes.

For many years, various drugs have been tested for their ability toalter the expression of genes or the translation of their messages intoprotein products. One problem with existing drug therapy is that ittends to act indiscriminately and affect healthy cells as well asneoplastic cells. This is a major problem with many forms ofchemotherapy where there are severe side effects primarily due to theaction of the toxic drugs on healthy cells.

In view of the foregoing, there remains a need for therapeutic agentswhich specifically inhibit the over expression of genes associated withcell-proliferative disorders, but have limited negative effects onhealthy cells.

SUMMARY OF THE INVENTION

The initial response of a cell to an external stimulus results in theinduction of a select set of genes, many of which are nuclearproto-oncogenes. Prominent among the products of these early responsegenes are Fos and Jun which serve as a paradigm for cooperation betweenoncoproteins to activate transcription of specific promoters. The Fosand Jun proteins bind to AP-1 sites as heterodimers. The heterodimericcomplex activates transcription of genes containing AP-1 binding sites.

During study of the FosB gene, a surprising means of regulatingtranscription was discovered. It was found that a truncation of FosB,which occurs by deletion of a region of an exon which encodes atrans-activation domain, results in a trans-repressing protein whichcauses suppression of promoters containing the AP-1 site.

In a first aspect, the present invention relates to a polynucleotideencoding a trans-repressing protein of the Fos gene family characterizedas encoding a protein (1) having a leucine zipper domain, and (2)forming a heterodimer with a Jun related protein, wherein theheterodimer is capable of binding to an AP-1 site and suppressingtranscriptional transactivation of a promoter containing the AP-1 site.

In a second aspect, the present invention relates to a trans-repressingprotein encoded by polynucleotide of the invention.

In a further aspect, the present invention relates to a method oftreating a cell proliferative disorder wherein the disorder isassociated with activation of a promoter containing an AP-1 site whichcomprises (1) removing a sample of tissue associated with the disorderfrom a subject with the disorder, (2) isolating from the tissue sample ahematopoietic cell which has infiltrated the tissue, (3) contacting thehematopoietic cell with a recombinant expression vector comprising apolynucleotide encoding the Fos protein of the invention, and (4)introducing the hematopoietic cell into the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b shows the structure of FosB2 coding sequence. In (A),FosB2 coding region is aligned with FosB cDNA. The entire codingsequence from nucleotide 1202 to 2218 (based on numbering system inZerial, et al., EMBO J., 8:805, 1989) including the intron-derived exonfrom 1913 to 2052 (the hatched box), is shown. The stop codon (TGA) inFosB2 reading frame is indicated. BR box represents the basic region,and L--L--L--L--L box indicates the "leucine zipper". In FIG. 1(B), thesequence of the alternative spliced intron of FosB gene is shown.Nucleotide sequence of mouse genomic DNA surrounding the alternativeintron of FosB gene is shown in the top line. FosB and FosB2 cDNAsequences are aligned and presented in the middle line and bottom line,respectively. The conserved splicing donor (GT) and acceptor (AG) areboldfaced. The consensus branch point sequence (CTGAC) and a pyrimidinestretch which is involved in lariat formation are underlined. The stopcodon (TGA) of FosB2 cDNA is also underlined, (...) indicates completeidentity.

FIG. 2A and 2B shows mouse FosB DNA and deduced amino acid sequences.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Jun and Fos proteins play an important role in the regulation of geneexpression in .many cell types. Known Jun proteins include c-Jun, JunBand JunD. Fos and Jun proteins contain a distinctive "leucine zipper"domain which enables members of both families to form heterodimers andsubsequently bind to AP-1 sites. The heterodimer, once bound, activatestranscriptional transactivation of a promoter containing an AP-1 site.Cooperation between these two nuclear oncoproteins is required forefficient transcriptional activation of the gene associated with theAP-1 site.

In contrast to the foregoing, the present invention relates to a newmember of the Fos gene family which is a trans-repressing proteindesignated FosB2. This protein is a truncated form of FosB. AlthoughFosB2 associates with a Jun protein to form heterodimers which bind toAP-1 sites, these FosB2 containing heterdoimers are unable totransactivate transcription.

FosB2, is a trans-acting molecule which has a negative regulatoryactivity. A trans-activating locus commonly refers to a DNA sequencewhich encodes a molecule that affects another DNA sequence located on achromosome different from the trans-activating locus. Instead offunctioning to activate transcription at a particular site in the DNA,FosB2 acts to negatively affect transcription, perhaps by sequesteringthe Jun protein and thereby interfering with positive regulatoryelements.

Other proteins regulating gene expression have been found whichapparently "zip" together in pairs. The formation of this stable complexis required before the proteins can effectively bind to a specificregion of DNA. The area of the zipper where the proteins associate isrich in periodically spaced leucine residues, thus the term "leucinezipper". Regions of certain proteins contain a leucine residue at everyseventh position of an alpha helix which, through hydrophobicinteractions, enables the proteins to form dimers. The trans-repressingFosB2 described in the present invention contains a classical leucinezipper domain. This allows FosB2 to form a complex with proteins of theJun family, which also contain a leucine zipper domain. This leucinezipper structure is found in various transcription factors such as Fos,Myc, and Jun; the yeast transcription factors GCN4 and yAP-1, enhancerbinding protein (C/EBP), and cAMP responsive element-binding protein(CREB). The FosB2 protein of the present invention associates with knownJun proteins encoded by c-Jun, JunB and JunD, however, FosB2 can alsoassociate with other proteins containing a Jun related leucine zipper tonegatively regulate transcription.

Association of FosB2 with members of the Jun family or other proteincontaining a Jun related leucine zipper is necessary for binding to DNA.While the trans-repressing protein of the invention, complexed with amember of the Jun family, preferably binds to an AP-1 site, it mayalternatively bind to another site containing the consensus sequencefound within AP-1.

Genomic DNA encoding FosB2 protein contains interrupting sequences(introns) and coding sequences (exons). The truncation event whichresults in FosB2 is caused by a deletion of 140 base pairs from an exonof the FosB gene. This deletion of coding sequence from nucleotides 1913through 2052 results from alternative splicing wherein the entire FosBgene is initially transcribed into one entire mRNA which is subsequentlycut such that only RNA sequences on either side of the cut remain. As aresult, the biological function of the FosB gene is expanded by thesplicing event. In FosB2, an exon encoding a trans-activation domain isspliced out of FosB mRNA, thereby creating FosB2 which lackstrans-activating activity. However, although unable to transactivatetranscription, FosB2 retains both the DNA binding and the leucine zipperdomain and, as such, can still complex with Jun family proteins. TheseFosB2/Jun complexes can still bind to promoters with AP-1 sites, but areunable to transactivate the cell. Consequently, FosB2 acts as aregulatory factor by negatively affecting the transcription of genescontaining AP-1 sites. In addition, FosB2 interferes with thetransforming ability of FosB and other members of the Fos gene family bycompeting with Jun proteins in the formation of FosB/Jun complexes.

The invention provides polynucleotides encoding the FosB2trans-repressing protein. These polynucleotides include DNA, cDNA andRNA sequences which encode transrepressing protein. It is understoodthat all polynucleotides encoding all or a portion of FosB2 are alsoincluded herein, so long as they exhibit the trans-repressing activityof FosB2. Such polynucleotides include both naturally occurring andintentionally manipulated, for example, mutagenized polynucleotides.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences and 2) antibody screening of expression librariesto detect shared structural features.

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

A FosB2 cDNA library can be screened by injecting the various cDNAs intooocytes, allowing sufficient time for expression of the cDNA geneproducts to occur, and testing for the presence of the desired cDNAexpression product, for example, by using antibody specific for FosB2polypeptide or by using functional assays for FosB2 binding activity.

Alternatively, a cDNA library can be screened indirectly for FosB2peptides having at least one epitope using antibodies to FosB2. Suchantibodies can be either polyclonally or monoclonally derived and usedto detect expression product indicative of the presence of FosB2 cDNA.

The development of specific DNA sequences encoding FosB2 can also beobtained by: (1) isolation of a double-stranded DNA sequence from thegenomic DNA; (2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and (3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the use of genomic DNA isolates (1),is the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides because of thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the formation of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNNDNA hybridization procedures which are carried outon cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucleic Acid Research, 11:2325,1983).

DNA sequences encoding FosB2 can be expressed in vitro by DNA transferinto a suitable host cell. "Recombinant host cells" or "host cells" arecells in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that not all progeny are identical to the parental cell sincethere may be mutations that occur at replication. However, such progenyare included when the terms above are used.

Host cells may also include yeast. cDNA can be expressed in yeast byinserting the cDNA into appropriate expression vectors and introducingthe product into the host cells. Various shuttle vectors for theexpression of foreign genes in yeast have been reported (Heinemann, etal., Nature, 340:205, 1989; Rose, et al., Gene, 60:237, 1987). DNAsequences encoding FosB2 can be expressed in vivo in either prokaryotesor eukaryotes. Methods of expressing DNA sequences having eukaryoticcoding sequences in prokaryotes are well known in the art. Biologicallyfunctional virai and plasmid DNA vectors capable of expression andreplication in a host are known in the art. Such vectors are used toincorporate DNA sequences of the invention. Hosts include microbial,yeast and mammalian organisms.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, insertion of a plasmid encased in liposomes, orvirus vectors may be used. Eukaryotic cells can also be cotransformedwith foreign cDNA encoding FosB2 protein, and a second foreign DNAmolecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express theprotein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982).

Isolation and purification of microbially expressed protein, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

Antibodies provided in the present invention are immunoreactive withFosB2 protein. Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et a., Nature, 256:495, 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989).

Minor modifications of FosB2 primary amino acid sequence may result inproteins which have substantially equivalent activity compared to theFosB2 proteins described herein. Such modifications may be deliberate,as by site-directed mutagenesis, or may be-spontaneous. All proteinsproduced by these modifications are included herein as long as FosB2activity exists.

In the present invention, the FosB2 sequences may be inserted into arecombinant expression vector. The term "recombinant expression vector"refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of FosB2 geneticsequences. Such expression vectors contain a promotor sequence whichfacilitates the efficient transcription of the inserted genetic sequencein the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders which are mediated by Fos/Jun proteincomplexes. Such therapy would achieve its therapeutic effect byintroduction of the FosB2 polynucleotide, into cells of animals havingthe proliferative disorder. Delivery of FosB2 polynucleotide can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system.

The term "cell-proliferative disorder" denotes malignant as well asnonmalignant cell populations which morphologically often appear todiffer from the surrounding tissue. For example, the FosB2polynucleotide is useful in treating malignancies of the various organsystems, such as, for example, lung, breast, lymphoid, gastrointestinal,and genito-urinary tract as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer, non-small cell carcinoma of the lung, cancer of the smallintestine, and cancer of the esophagus. The FosB2 polynucleotide is alsouseful in treating non-malignant cell-proliferative diseases such aspsoriasis, pemphigus vulgaris, Behcet's syndrome, and lipidhistiocytosis. Essentially, any disorder which is etiologically linkedto the formation of the Fos/Jun family heterodimer would be consideredsusceptible to treatment with FosB2 polynucleotide.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), HaNey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retrovirai vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a FosB2 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the FosB2 polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA 12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retrovital virions.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for FosB2 polynucleotides a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelies, mixed micelies, andliposomes. The preferred colloidal system of this invention is aliposome. Uposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoyiphosphatidyicholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganeile-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and "home in" on the desired cells. A ligand maybe any compound of interest which will bind to another compound, such asa receptor.

In general, surface membrane proteins which bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies are preferred receptors. Antibodies can be used to targetliposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), may be exploited for the purpose oftargeting antibody-FosB2-containing liposomes directly to the malignanttumor. Since the FosB2 gene product may be indiscriminate with respectto cell type in its action, a targeted delivery system offers asignificant improvement over randomly injecting nonspecific liposomes. Anumber of procedures can be used to covalently attach either polyclonalor monoclonal antibodies to a liposome bilayer. Antibody-targetedliposomes can include monoclonal or polyclonal antibodies or fragmentsthereof such as Fab, or F(ab')₂, as long as they bind efficiently to anthe antigenic epitope on the target cells. Uposomes may also be targetedto cells expressing receptors for hormones or other serum factors.

The invention also provides a method for suppressing the transcriptionaltransactivation of a promoter containing an AP-1 site by contacting theAP-1 site with an effective amount of the trans-repressing protein. Aneffective amount is meant to include that level which results in thedeactivation of a previously activated promoter containing AP-1. Theamount of FosB2 transrepressing protein required would be that amountnecessary to displace Fos protein in an existing Fos/Jun complex in thecell, or that amount necessary to compete with Fos protein to formcomplexes with the various Jun proteins. By functionally inactivatingthe Fos/Jun complex, both transcription and transformation can besuppressed. Delivery of an effective amount of the trans-repressingprotein can be accomplished by one of the mechanisms previouslydescribed, such as by retroviral vectors or liposomes, or other methodswell known in the art.

In addition, the invention discloses a method of treating cellproliferative disorders, by removal of a tissue sample from a subjectwith the disorder; isolating hematopoietic cells from the tissue sample;and contacting isolated cells with a recombinant expression vectorcontaining DNA encoding FosB2 protein and, optionally, a target specificgene. Optionally, the cells can be treated with a growth factor, such asinterleukin-2, to stimulate cell growth, before reintroducing the cellsinto the subject. When reintroduced, the cells will specifically targetthe cell population from which they were originally isolated. In thisway, the trans-repressing activity of FosB2 may be used to inhibitundesirable cell proliferation in a subject.

An alternative use for recombinant retroviral vectors comprises theintroduction of polynucleotide sequences into the host by means of skintransplants of cells containing the virus. Long term expression offoreign genes in implants, using cells of fibroblast origin, may beachieved if a strong housekeeping gene promoter is used to drivetranscription. For example, the dihydrofolate reductase (DHFR) genepromoter may be used. Cells such as fibroblasts, can be infected withvirions containing a retroviral construct containing the gene ofinterest, for example FosB2, together with a gene which allows forspecific targeting, such as TAA, and a strong promoter. The infectedcells can be embedded in a collagen matrix which can be grafted into theconnective tissue of the dermis in the recipient subject. As theretrovirus proliferates and escapes the matrix it will specificallyinfect the target cell population. In this way the transplantationresults in increased amounts of trans-repressing FosB2 being produced incells manifesting the cell proliferative disorder.

Because the present invention identifies a nucleotide sequence expressedin FosB, but not in FosB2, it is possible to design appropriatetherapeutic or diagnostic techniques directed to this unique sequence ofFosB. Thus, where a cell-proliferative disorder is associated with theexpression of FosB, the unique sequence associated with FosB, but notwith FosB2, could be used to produce nucleic acid sequences thatinterfere with FosB expression at the translational level. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific FosB mRNA, either by masking that mRNA with anantisense nucleic acid or by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNAsince the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target FosB-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while "hammerhead"-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

Antisense sequences can be therapeutically administered by techniques asthose described above for the administration of FosB2 polynucleotides.Especially preferred for therapeutic delivery of antisense sequences isthe use of targeted liposomes.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 FosB2 cDNA CLONING

FosB2 cDNA which contains the entire coding sequence was obtained duringcloning of the FosB cDNA by polymerase chain reaction (PCR) (Sherman, etal., TIG, 5:137, 1989). Primers corresponding to nucleotides (NT)1284-1301 and 2212-2227 of the published sequence (Zerial, et al., EMBOJ., 8:805-813, 1989) were used to clone FosB cDNAs using PCR. Multipleindependent clones were selected and analyzed by nucleotide sequencing.

Characterization of FosB2 cDNA

During the process of molecular cloning of FosB cDNA by PCR usingoligonucleotides based on the published FosB sequence (Zerial, et al.,EMBO J., 8:805-813, 1989), DNA fragments of approximately 0.9 to 1.0 kbpwere obtained. These cDNA fragments were subcloned and when thenucleotide sequence of the fragments was determined, it became apparentthat many of them were missing 140 bp as compared to previouslydescribed FosB cDNAs. More importantly, the open reading frame of theshorter cDNA could encode a protein of only 237 aa as compared to the338 aa protein encoded by the FosB cDNA.

FIG. 1 schematically illustrates the molecular structure of both theFosB and the shorter cDNA termed FosB2. The entire coding sequence fromnucleotide 1202 to 2218 (based on numbering system in Zerial, et al.,supra) including the intron-derived exon from 1913 to 2052 (the hatchedbox), is shown.

The nucleotide and deduced amino acid sequence of the first 237 aa ofthe two proteins are identical (Zerial, et al., EMBO J., 8:805-813,1989). Examination of the sequences of FosB and FosB2 cDNA revealed thatthe FosB2 protein terminates immediately following the 140 bp deletion.Thus, the remaining sequence in the 3' region of FosB2 is identical toFosB but cannot be translated in this frame. The FosB2 protein, however,does contain the leucine zipper domain (aa 183 to 211) and the basicregion (aa 157 to 182), which is well conserved among all members of theFos family (Matsui, et al., Oncogene, 5:249-255, 1990).

The nucleotide sequence of a corresponding FosB genomic DNA fragment wasdetermined to address the question of the nature of the 140 bp insert inFosB. A comparison of the sequences of FosB genomic DNA, and FosB andFosB2 cDNAs revealed that the 140 bp insert has all the hallmarks of abonafide type II intron. In addition to the consensus splice donor(AG↓GTGAGA) and acceptor (CAG) sites, there are potential branch points23 and 28 NT from the AG and a pyrimidine-rich stretch between branchpoint and splice site. Because the 140 bp intron is deleted in FosB2,the reading frame shifts by -1, creating the stop codon TGA. Therefore,FosB2, the product of spliced FosB mRNA, is only 237 aa long.

EXAMPLE 2 PLASMID DNA CONSTRUCTION AND RNase PROTECTION ANALYSIS

Plasmid SK-FosB2 contains the entire FosB coding sequence except theintron-derived exon, in the vector SK. Plasmid SK-FosB contains theentire FosB coding sequence. pBKFosB and pBKFosB2 are expressionconstructs of FosB and FosB2 in the FBJ-LTR driven expression vectorpBKPL. The analogous c-Fos expression plasmid pBK28, and the c-junexpression plasmid pSV-c-jun have been described (Lamph, et al., Nature,334:629-631, 1988; Sassone-Corsi, et al., Cell, 54:553-560, 1988).

Southern blotting analysis of genomic DNA suggested the presence of asingle FosB gene. RNA was prepared from cells induced with serum bymethods known in the art, and hybridized to FosB2 probe. Within 60minutes following serum induction, maximal levels of a 5.0 kb species ofRNA was detected. Because the alternative spliced form of FosB RNA isshorter by 140 bp, the analysis performed here was not able todistinguish between the two forms of RNA. To confirm the existence ofalternatively spliced forms of FosB mRNAs, RNase protection analysis wasperformed using probes generated from either FosB2 or FosB cDNA. Uponannealing with full length FosB mRNA and subsequent RNase digestion, a166 NT fragment diagnostic for FosB RNA was observed. Other probes usedin RNase protection assays included a FosB Ncol fragment which is 835NT. This probe protected the expected 784 NT FosB fragment. In addition,the same probe yielded 478 NT and 166 NT bands, representative of FosB2and the 140 bp deletion.

Five μg of cytoplasmic RNA were subjected to RNA blot analysis. FosB2coding region of cDNA was used as the hybridization probe. Uniformly ³²P-UTP-labeled complementary RNA probes were synthesized. Results showedcoordinated regulation of FosB and FosB2 mRNA by serum in the absenceand presence of cyclohexamide in NIH 3T3 fibroblasts.

For precise quantitation of the abundance of two species of RNAs,analysis was performed using probes generated from FosB cDNA. In cellsstimulated with serum, appropriate length FosB and FosB2 specificfragments were identified. From densitometric scanning of the bandintensity, length of fragments, and uracil content of fragments, itappeared that FosB2 was slightly more abundant (FosB/FosB2=) than FosBmRNA.

The results indicated that (i) bands of the expected sizes correspondingto FosB and FosB2 RNA were identified, (ii) FosB and FosB2 expressionwas stimulated by serum and (iii) induction of FosB and FosB2 mRNA byserum was superinduced with cycloheximide. No induction of FosB or FosB2mRNA was detected when the cells were treated with tetradecanoyl phorbol13-acetate (TPA). FosB is thus unique among early response genes in thatit is inducible with serum, but not TPA.

EXAMPLE 3 PROTEIN ANALYSIS BY IMMUNOPRECIPITATION

NIH 3T3 cells were starved with 0.5% bovine calf serum for 2 days, andthen stimulated with 10% dialyzed calf serum in the presence ofmethionine- and cysteine-free media. Forty minutes before harvest, thecells were labeled with ³⁵ S-methionine (200 μCi/ml and cell lysateswere prepared (Barber, et al., Mol. Cell. Biol., 7:2201-2211, 1987).Lysates containing equal amounts of protein were used forimmunoprecipitation with anti-FosB specific antibody 5108-1B (againstFosB peptide residues from 80 to 97) or a c-Fos monoclonal antibody18H6.

Several extensively phosphorylated forms of c-Fos protein wereimmunoprecipitated with 18H6. Maximum levels of precipitated proteinwere obtained between 30-60 minutes following serum induction and amarked decrease was observed at 120 minutes. No Jun related proteinswere identified since the extracts were boiled prior toimmunoprecipitation which disrupted the Fos-Jun complex. In parallelexperiments, 5108-1B antiserum immunoprecipitated three polypeptides ofapproximate molecular weight of 65 kD, 55 kD, and 43 kD, after seruminduction. These polypeptides were identified in quiescent cells, butthe levels increased by 40 minutes and declined to basal levels by90-120 minutes following serum induction. When the antibody waspreincubated with synthetic peptide against which the antisera wasraised, two of the polypeptides were not detected. On the other hand,the immunoprecipitation of these two polypeptides was not affected ifthe antisera were preincubated with a non-specific peptide (aa 152 to176 of murine c-Rel) originating from c-Rel protein. Neither the 65 or55 kD polypeptides was observed with the pre-immune sera. Since there isnot an antisera which distinguishes between FosB and FosB2 at present,it cannot be determined as to which of the two polypeptides is theproduct of FosB or FosB2 RNAs. However, both polypeptides, as well as invitro translated FosB protein, show common peptides following digestionwith V8 protease. From the molecular weights of the in vitro translatedFosB and FosB 2 RNA transcripts and the possible posttranslationalmodifications, the 65 kD polypeptide is FosB and the 55 kD polypeptideFos B2. The lower band was non-specifically precipitated by protein Asepharose and was not blocked by FosB peptide. It is likely that thislowest molecular weight band is actin, which is inducible with serum andmigrates at a position of about 43 kD.

EXAMPLE 4 IN VITRO TRANSCRIPTION AND TRANSLATION

T7-c-Fos, pGEM-c-Jun, SK-FosB and SK-FosB2 were all linearized withEcoRI and transcribed with T7 polymerase or T3 polymerase. Each RNA wastranslated with micrococcal nuclease-treated, methionine-free rabbitreticulocyte lysate as directed by the supplier (Promega, Madison, WI).For gel shift assays, equal molar amounts of unlabeled proteins werecombined, and incubated with ³² P-γTP labeled TRE/AP-1 oligonucleotideat room temperature for 15 minutes and the binding complex formation wasanalyzed on a native 4% acrylamide gel (Sassone-Corsi, et al., Nature,336:692-695, 1988; Dwarki, et al., EMBO J., 9:225,232, 1990).

FosB2 binds to AP-1 site

A hallmark of the members of the Fos gene family is that their productsform heterodimers with members of Jun family and bind to AP-1 sites. Invitro translated FosB2 and c-Jun was found to bind efficiently to anAP-1 site. Competition with excess unlabeled oligonucleotide containingthe cognate AP-1 site abolished binding. Little or no binding wasobserved with either FosB2, FosB, c-Fos or c-Jun alone. To confirm thatDNA binding by the FosB2-c-Jun complex was dependent on heterodimerformation via the leucine zipper domain, RNAs encoding FosB2 and a c-Junmutant containing a deletion of the leucine zipper were cotranslated invitro (Ransone, et al., Genes Dev., 3:770-781, 1989). No binding to theAP-1 site was detected.

FosB2 can efficiently bind to an AP-1 site in association with c-Jun.Various combinations of expression plasmids for FosB, FosB2, and c-Jun(pBKFosB, pBKFosB2, pSVc-jun and pBK28) along with a reporter plasmid5XTRE-TKCAT, which contains five copies of the TPA-responsive element(TRE) linked to a CAT reporter gene, were cotransfected into F9teratocarcinoma cells to determine whether FosB2 in association with Juncould cause transcriptional transactivation of promoters containing AP-1binding sites. Briefly, embryonic carcinoma F9 cells were plated in 10cm dishes at a density of 5×10⁶ cells/plate 24 hours beforetransfection. Transfection protocol was as described (Chen, et al., Mol.Cell. Biol., 7:2745-2752, 1987) and β-galactosidase activity was assayedby the CNPG reaction (Nielsen, et al., Proc. Natl. Acad. Sci. USA,80:5198-5202, 1983). Two μg of the reporter construct 5XTRECAT and 2 μgof pBAG (a β-galactosidase expression plasmid, which served as aninternal control of transfection efficiency) were cotransfected into F9cells with various combinations of expression plasmids.

For CAT assays, cell extracts containing equal β-GAL activity wereincubated with ¹⁴ C-chloramphenicol, and the reaction products wereanalyzed on TLC plates as described (Gorman, et al., Mol. Cell. Biol.,2:1044-1051, 1982). Forty-eight hours after transfection, CAT activitywas measured. All CAT activity assays were standardized withβ-galactosidase activity. Fold induction was standardized with thecontrol reaction (no expression plasmid) and three independentexperiments were performed. c-Jun (0.5 μg) was cotransfected with 0-12μg of c-Fos, FosB, or FosB2 expression plasmid.

Like c-Fos, FosB was able to transactivate transcription in cooperationwith c-Jun. FosB2 on the other hand was unable to transactivatetranscription. Because FosB2 can form heterodimers with c-Jun and bindto AP-1 site, it apparently interferes with transcriptional activationby c-Fos or FosB proteins. Upon increasing the concentration oftransfected FosB2 plasmid, the transactivation by c-Fos wasincrementally reduced. FosB2 also appeared to suppress transcriptionaltransactivation by FosB. When the amount of c-Fos DNA was increased to12 μg or addition of 6 μg of FosB DNA, instead of FosB2 DNA, CATactivity increased. This indicated that reduced CAT activity uponaddition of FosB2 plasmid was not due to adventitious events likesquelching. Furthermore, immunostaining of the transfected cells withc-Fos monoclonal antibody (18H6) showed that c-Fos protein wassynthesized. Finally, FosB2 mutants in the leucine zipper domain wereunable to suppress transactivation by c-Fos and c-Jun. Thus, the resultsshow that FosB2 can interfere with transcriptional transactivation byFos proteins such as c-Fos and FosB.

EXAMPLE 5 FosB2 SUPPRESSES TRANSFORMATION BY Fos PROTEINS

Increasing amounts of FosB2 plasmid decreased the transforming potentialof v-Fos, c-Fos, and FosB proteins, as seen in Table 1. In agreementwith previous results, v-Fos is a more potent transforming agent thanits cellular cognate, c-Fos (Miller, et al., Cell, 36:51-60, 1984).Surprisingly, however, FosB appeared to have a transforming potentialequivalent to that of the v-Fos gene. These results also emphasize alink between transactivation potential and cellular transformation byFos proteins.

208F cells were transfected with 2 μg of transforming plasmid DNA(v-Fos, c-Fos, or FosB) along with different amounts of a FosB2expression plasmid (from 0-16 μg); the total amount of DNA used in eachtransfection was kept constant by varying the amount of carrier DNA.Focus assays were performed as previously described (Miller, et al.,Cell, 36:51-60, 1984). Each transfection was plated in duplicate. Fociwere counted 12 days after transfection for v-Fos transfected cells, and17 days after transfection for c-Fos and FosB transfected cells. FosB2interfered with both the transcriptional transactivation andtransformation potential of c-Fos and FosB, suggesting a role as atrans-negative regulator.

                  TABLE 1                                                         ______________________________________                                                                          Transformation                              Transforming                                                                            FosB2        Number of Foci                                                                           Efficiency                                  DNA (2 μg)                                                                           (μg) Expt. 1   Expt. 2                                                                             (Average)                                   ______________________________________                                        v-Fos     0       130/108   55/67 100                                                   2       58/75     30/48 60                                                    16      36/38      9/15 25                                          c-Fos     0       43/31     21/26 100                                                   2       16/8      5/3   17                                                    16      2/8       5/2   15                                          FosB      0       163/175   48/61 100                                                   2       102/82    43/25 51                                                    16      38/36     28/17 31                                          None      16      0/0       0/0                                               ______________________________________                                    

EXAMPLE 6 IN VIVO EXPRESSION OF RETROVIRAL MEDIATED GENE TRANSFER INIMPLANTS

Although the following example is directed to chimeric retroviralexpression in mice, the techniques described herein are readily modifiedfor use with other species, such as humans. In practice, the retroviralvectors of the invention containing FosB2 polynucleotide would furthercontain a gene which would express a binding protein or some otherligand which, upon release of the retrovirus from the implant, wouldallow the retrovirus to specifically target a particular cell type.Those of skill in the art can readily make such modifications withoutresorting to undue experimentation.

Animal and cell culture Conditions

Adult male C57 BL/6J mice (6-8 weeks old) and Nu/Nu athymic mice can beobtained from the Jackson Laboratory, ME. The retroviral packaging celllines Ψ-CRE and Ψ-CRIP (Danos, et al., Proc. Natl. Acad. Sci. USA,85:6460-6464, 1988) and the cell lines NIH 3T3 and rat 208F aremaintained in Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith 10% bovine calf serum. Primary fibroblasts are obtained from day 17embryos of C57 BL/6J mice and are grown in DMEM supplemented with 10%fetal calf serum. Infected cells are then selected in medium containing400 μg/ml of G418.

Vector Construction

Retroviral vectors such as LNCdF9L can be used for the vectorconstruction (Axelrod, et al., Proc. Natl. Acad. Sci., 87:5173-6177,1990). The vectors are generated by inserting a 3.1 kBP Barn H1 fragmentcontaining the entire coding sequence of the β-galactosidase gene, forexample, into the Bgl II site of plasmid LNL-SLX to generate the vectorLNL-SLXβgal. The LNL-SLX vector is a derivative of LNL-XHC (Bender, etal., J. Virology, 61:1639-1646, 1987) and contains a new polylinker toincrease the number of cloning sites. A 350 bp Hind III fragment of themouse dihydrofolate reductase (DHFR) promoter can be cloned in theunique Hind III site of LNL-SLXβgal. A Bam HI/Hind III fragmentcontaining the human intermediate early cytomegalovirus (CMV-IE)promoter (-522 to +55; Nelson, et al., Mol. Cell. Biol., 7:4125-4129,1987) can be cloned in the Bam HI/Hindlll site of LNL-SLXβgal. The geneof interest (FosB2), along with a target-specific gene, can be clonedinto the vector to generate target-specific vehicles for the FosB2 geneand its gene product.

Analysis of β-galactosidase activity

Beta-galactosidase histochemistry is performed according to Sanes, etal. (EMBO J., 5:3133, 1986) with minor modifications. Cultured cells arerinsed with phosphate buffered saline solution (PBS) pH 7.4 and thenfixed for 5 minutes on ice in 2% formaldehyde plus 0.2% glutaraldehydein PBS. The cells are then rinsed 2 times with PBS and overlaid with asolution containing 1 mg/mi 4-Cl-5-Br-3-indodyl-β-galactosidase (X-gal),5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl₂in PBS pH 7.4. Incubation is performed at 37° C. for 2 to 24 hours. Toanalyze β-galactosidase activity in the artificial collagen matrix, thefixation is prolonged for 30 minutes on ice.

Virus production

Ten μg of plasmid vector DNA is transfected into the ecotropic packagingcell line Ψ-CRE by the calcium phosphate co-precipitation method. Otherpackaging cell lines known in the art can alternately be used. Themedium is changed 24 hours later and 48 hours after transfection, theculture medium is harvested and used to infect the amphotropic packagingcell line Ψ-CRIP, for example, in the presence of 8 μg/ml of polybrene.Single colonies of infected Ψ-CRIP cells are isolated by selection inthe presence of G418-containing medium and expanded. Recombinantretroviruses are harvested from confluent culture dishes, filtered, andused to infect mouse embryo fibroblasts cells in the presence ofpolybrene to determine the viral titers. Twenty-four hours afterinfection, the medium is changed to G418 containing medium and coloniesstained and counted after 12 to 14 days. The presence of helper virus isassayed by the marker residue method (Keller, et al., Nature,318:149-154, 1985). Briefly, the medium from the infected cells is usedto infect naive NIH 3T3 cells. The presence of β-galactosidase positivecells is determined after 72 hours and the presence of G418 resistantcolonies is quantified after 14 days.

Implantation of infected mouse embryo fibroblasts in mice

Infected mouse embryo fibroblast cells are embedded in a collagen matrixas previously described (Louis, et al., Proc. Natl. Acad. Sci. USA,85:3150-3154, 1988). The collagen matrix containing 2×10⁶ infectedfibroblasts is then grafted into the connective tissue of the dermis inthe mid-back of recipient mice. To ensure rapid vascularization of thegrafted tissue, a 2 mm² piece of gelfoam (Upjohn) containing 24 g ofbasic fibroblast growth factor is inserted into the connective tissuealong with each graft (Louis, et al., id.) At different intervals oftime, the implanted artificial collagen matrix is removed and stainedfor β-galactosidase activity.

Use of housekeeping gene promoters

Sustained expression in the implants may be a function of the type ofthe promoter used to initiate the transcription of the foreign gene.Since CMV is an inducible promoter and may require actively growingcells, a promoter which maintains the constitutive levels of manyhousekeeping genes is used. Retroviral vectors containing murinedihydrofolate reductase gene promoter, for example, and the bacterialβ-galactosidase gene as a reporter are used. Clones producing high titreamphotropic recombinant viruses are selected by infecting NIH 3T3 cells,and analyzing for β-galactosidase activity and the presence of helperviruses as described in this example.

Further characterization of the recombinant viruses can be accomplishedby analysis of the RNA transcripts from cells infected with the LNL-SLXβ-gal viruses. Transcripts of the expected size will be detectable invirus-producing CRIP cells or mouse embryo fibroblasts. No transcriptswill be detected in uninfected cells.

β-galactosidase expression in mice

Mouse embryo fibroblasts are infected with either LNL-SLX CMV β-gal orLNL-SLX DHFR β-gal viruses to test if the sustained expression ofβ-galactosidase can be attained in vivo. Infected cells are embedded ina collagen matrix and grafted in mice. After different time intervals,the grafts are explanted and analyzed for the presence ofβ-galactosidase positive cells by staining blue with X-Gal. A minimum oftwo to three grafts are explanted at each time point.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence (and the deduced aminoacid sequence) of a genomic fragment encoding a mouse-derived FosBprotein.

Sequence ID No. 2 is the deduced amino acid sequence of a mouse-derivedFosB protein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4144 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: MouseFosB                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1202..2216                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATAAATTCTTATTTTGACACTCACCAAAATAGTCACCTGGAAAACCCGCTTTTTGTGACA60                AAGTACAGAAGGCTTGGTCACATTTAAATCACTGAG AACTAGAGAGAAATACTATCGCAA120              ACTGTAATAGACATTACATCCATAAAAGTTTCCCCAGTCCTTATTGTAATATTGCACAGT180               GCAATTGCTACATGGCAAACTAGTGTAGCATAGAAGTCAAAGCAAAAACAAACCAAAGAA240               AGGAGCCACA AGAGTAAAACTGTTCAACAGTTAATAGTTCAAACTAAGCCATTGAATCTA300              TCATTGGGATCGTTAAAATGAATCTTCCTACACCTTGCAGTGTATGATTTAACTTTTACA360               GAACACAAGCCAAGTTTAAAATCAGCAGTAGAGATATTAAAATGAAAAGGTTTG CTAATA420              GAGTAACATTAAATACCCTGAAGGAAAAAAAACCTAAATATCAAAATAACTGATTAAAAT480               TCACTTGCAAATTAGCACACGAATATGCAACTTGGAAATCATGCAGTGTTTTATTTAAGA540               AAACATAAAACAAAACTATTAAAATAGTT TTAGAGGGGGTAAAATCCAGGTCCTCTGCCA600              GGATGCTAAAATTAGACTTCAGGGGAATTTTGAAGTCTTCAATTTTGAAACCTATTAAAA660               AGCCCATGATTACAGTTAATTAAGAGCAGTGCACGCAACAGTGACACGCCTTTAGAGAGC720               ATT ACTGTGTATGAACATGTTGGCTGCTACCAGCCACAGTCAATTTAACAAGGCTGCTCA780              GTCATGAACTTAATACAGAGAGAGCACGCCTAGGCAGCAAGCACAGCTTGCTGGGCCACT840               TTCCTCCCTGTCGTGACACAATCAATCCGTGTACTTGGTGTATCTGA AGCGCACGCTGCA900              CCGCGGCACTGCCCGGCGGGTTTCTGGGCGGGGAGCGATCCCCGCGTCGCCCCCCGTGAA960               ACCGACAGAGCCTGGACTTTCAGGAGGTACAGCGGCGGTCTGAAGGGGATCTGGGATCTT1020              GCAGAGGGAACTTGCATCGAA ACTTGGGCAGTTCTCCGAACCGGAGACTAAGCTTCCCCG1080             AGCAGCGCACTTTGGAGACGTGTCCGGTCTACTCCGGACTCGCATCTCATTCCACTCGGC1140              CATAGCCTTGGCTTCCCGGCGACCTCAGCGTGGTCACAGGGGCCCCCCTGTGCCCAGGGA120 0             AATGTTTCAAGCTTTTCCCGGAGACTACGACTCCGGCTCCCGGTGT1246                            MetPheGlnAlaPheProGlyAspTyrAspSerGlySerArgCys                                 1510 15                                                                       AGCTCATCACCCTCCGCCGAGTCTCAGTACCTGTCTTCGGTGGACTCC1294                          SerSerSerProSerAlaGluSerGlnTyrLeuSerSerValAspSer                              2025 30                                                                       TTCGGCAGTCCACCCACCGCCGCCGCCTCCCAGGAGTGCGCCGGTCTC1342                          PheGlySerProProThrAlaAlaAlaSerGlnGluCysAlaGlyLeu                              3540 45                                                                       GGGGAAATGCCCGGCTCCTTCGTGCCAACGGTCACCGCAATCACAACC1390                          GlyGluMetProGlySerPheValProThrValThrAlaIleThrThr                              5055 60                                                                       AGCCAGGATCTTCAGTGGCTCGTGCAACCCACCCTCATCTCTTCCATG1438                          SerGlnAspLeuGlnTrpLeuValGlnProThrLeuIleSerSerMet                              657075                                                                         GCCCAGTCCCAGGGGCAGCCACTGGCCTCCCAGCCTCCAGCTGTTGAC1486                         AlaGlnSerGlnGlyGlnProLeuAlaSerGlnProProAlaValAsp                              808590 95                                                                     CCTTATGACATGCCAGGAACCAGCTACTCAACCCCAGGCCTGAGTGCC1534                          ProTyrAspMetProGlyThrSerTyrSerThrProGlyLeuSerAla                              100105 110                                                                    TACAGCACTGGCGGGGCAAGCGGAAGTGGTGGGCCTTCAACCAGCACA1582                          TyrSerThrGlyGlyAlaSerGlySerGlyGlyProSerThrSerThr                              115120 125                                                                    ACCACCAGTGGACCTGTGTCTGCCCGTCCAGCCAGAGCCAGGCCTAGA1630                          ThrThrSerGlyProValSerAlaArgProAlaArgAlaArgProArg                              130135 140                                                                    AGACCCCGAGAAGAGACACTTACCCCAGAAGAAGAAGAAAAGCGAAGG1678                          ArgProArgGluGluThrLeuThrProGluGluGluGluLysArgArg                              145150155                                                                      GTTCGCAGAGAGCGGAACAAGCTGGCTGCAGCTAAGTGCAGGAACCGT1726                         ValArgArgGluArgAsnLysLeuAlaAlaAlaLysCysArgAsnArg                              160165170 175                                                                 CGGAGGGAGCTGACAGATCGACTTCAGGCGGAAACTGATCAGCTTGAA1774                          ArgArgGluLeuThrAspArgLeuGlnAlaGluThrAspGlnLeuGlu                              180185 190                                                                    GAGGAAAAGGCAGAGCTGGAGTCGGAGATCGCCGAGCTGCAAAAAGAG1822                          GluGluLysAlaGluLeuGluSerGluIleAlaGluLeuGlnLysGlu                              195200 205                                                                    AAGGAACGCCTGGAGTTTGTCCTGGTGGCCCACAAACCGGGCTGCAAG1870                          LysGluArgLeuGluPheValLeuValAlaHisLysProGlyCysLys                              210215 220                                                                    ATCCCCTACGAAGAGGGGCCGGGGCCAGGCCCGCTGGCCGAGGTGAGA1918                          IleProTyrGluGluGlyProGlyProGlyProLeuAlaGluValArg                              225230235                                                                      GATTTGCCAGGGTCAACATCCGCTAAGGAAGACGGCTTCGGCTGGCTG1966                         AspLeuProGlySerThrSerAlaLysGluAspGlyPheGlyTrpLeu                              240245250 255                                                                 CTGCCGCCCCCTCCACCACCCCCCCTGCCCTTCCAGAGCAGCCGAGAC2014                          LeuProProProProProProProLeuProPheGlnSerSerArgAsp                              260265 270                                                                    GCACCCCCCAACCTGACGGCTTCTCTCTTTACACACAGTGAAGTTCAA2062                          AlaProProAsnLeuThrAlaSerLeuPheThrHisSerGluValGln                              275280 285                                                                    GTCCTCGGCGACCCCTTCCCCGTTGTTAGCCCTTCGTACACTTCCTCG2110                          ValLeuGlyAspProPheProValValSerProSerTyrThrSerSer                              290295 300                                                                    TTTGTCCTCACCTGCCCGGAGGTCTCCGCGTTCGCCGGCGCCCAACGC2158                          PheValLeuThrCysProGluValSerAlaPheAlaGlyAlaGlnArg                              305310315                                                                      ACCAGCGGCAGCGAGCAGCCGTCCGACCCGCTGAACTCGCCCTCCCTT2206                         ThrSerGlySerGluGlnProSerAspProLeuAsnSerProSerLeu                              320325330 335                                                                 CTTGCTCTGTAAACTCTTTAGACAAACAAAACAAACAAACCCGCAAGGAA2256                        LeuAlaLeu                                                                     CAAGGAGGAGGAAGATGAGGAGGAGAGGGGAGGAAGCAGTCCGGGGGTGTGTGTGTGGAC2316              CCTTTGACTCTTCTGTCTGACCA CCTGCCGCCTCTGCCATCGGACATGACGGAAGGACCT2376             CCTTTGTGTTTTGTGCTCCGTCTCTGGTTTTCTGTGCCCCGGCGAGACCGGAGAGCTGGT2436              GACTTTGGGGACAGGGGGTGGGGCGGGGATGGACACCCCTCCTGCATATCTTTGTCCTGT2496              TACTTCAACCCAACTTCTGGGGATAGATGGCTGGCTGGGTGGGTAGGGTGGGGTGCAACG2556              CCCACCTTTGGCGTCTTGCGTGAGGCTGGAGGGGAAAGGGTGCTGAGTGTGGGGTGCAGG2616              GTGGGTTGAGGTCGAGCTGGCATGCACCTCCAGAGAGACCC AACGAGGAAATGACAGCAC2676             CGTCCTGTCCTTCTTTTCCCCCACCCACCCATCCACCCTCAAGGGTGCAGGGTGACCAAG2736              ATAGCTCTGTTTTGCTCCCTCGGGCCTTAGCTGATTAACTTAACATTTCCAAGAGGTTAC2796              AACCTCCTCCTGGACG AATTGAGCCCCCGACTGAGGGAAGTCGATGCCCCCTTTGGGAGT2856             CTGCTAACCCCACTTCCCGCTGATTCCAAAATGTGAACCCCTATCTGACTGCTCAGTCTT2916              TCCCTCCTGGGAAAACTGGCTCAGGTTGGATTTTTTTCCTCGTCTGCTACAGAGCCCCCT 2976             CCCAACTCAGGCCCGCTCCCACCCCTGTGCAGTATTATGCTATGTCCCTCTCACCCTCAC3036              CCCCACCCCAGGCGCCCTTGGCCGTCCTCGTTGGGCCTTACTGGTTTTGGGCAGCAGGGG3096              GCGCTGCGACGCCCATCTTGCTGGAGCGCTTTAT ACTGTGAATGAGTGGTCGGATTGCTG3156             GGTGCGCCGGATGGGATTGACCCCCAGCCCTCCAAAACTTTCCCTGGGCCTCCCCTTCTT3216              CCACTTGCTTCCTCCCTCCCCTTGACAGGGAGTTAGACTCGAAAGGATGACCACGACGCA3276              TCCCGGTGG CCTTCTTGCTCAGGCCCCAGACTTTTTCTCTTTAAGTCCTTCGCCTTCCCC3336             AGCCTAGGACGCCAACTTCTCCCCACCCTGGGAGCCCCGCATCCTCTCACAGAGGTCGAG3396              GCAATTTTCAGAGAAGTTTTCAGGGCTGAGGCTTTGGCTCCCCTATCCTCGA TATTTGAA3456             TCCCCAAATATTTTTGGACTAGCATACTTAAGAGGGGGCTGAGTTCCCACTATCCCACTC3516              CATCCAATTCCTTCAGTCCCAAAGACGAGTTCTGTCCCTTCCCTCCAGCTTTCACCTCGT3576              GAGAATCCCACGAGTCAGATTTCTATT TTTTAATATTGGGGAGATGGGCCCTACCGCCCG3636             TCCCCCGTGCTGCATGGAACATTCCATACCCTGTCCTGGGCCCTAGGTTCCAAACCTAAT3696              CCCAAACCCCACCCCCAGCTATTTATCCCTTTCCTGGTTCCCAAAAAGCACTTATATCTA3756              T TATGTATAAATAAATATATTATATATGAGTGTGCGTGTGTGTGCGTGTGCGTGCGTGCG3816             TGCGTGCGTGCGAGCTTCCTTGTTTTCAAGTGTGCTGTGGAGTTCAAAATCGCTTCTGGG3876              GATTTGAGTCAGACTTTCTGGCTGTCCCTTTTTGTCACCTTTTTG TTGTTGTCTCGGCTC3936             CTCTGGCTGTTGGAGACAGTCCCGGCCTCTCCCTTTATCCTTTCTCAAGTCTGTCTCGCT3996              CAGACCACTTCCAACATGTCTCCACTCTCAATGACTCTGATCTCCGGTTGTCTGTTAATT4056              CTGGATTTGTCGGGGACATG CAATTTTACTTCTGTAAGTAAGTGTGACTGGGTGGTAGAT4116             TTTTTACAATCTATATCGTTGAGAATTC4144                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 338 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetPheGlnAlaPheProGlyAspTyrAspSerGlySerArgCysSer                              151015                                                                        SerSerProSerAlaGlu SerGlnTyrLeuSerSerValAspSerPhe                             202530                                                                        GlySerProProThrAlaAlaAlaSerGlnGluCysAlaGlyLeuGly                              3540 45                                                                       GluMetProGlySerPheValProThrValThrAlaIleThrThrSer                              505560                                                                        GlnAspLeuGlnTrpLeuValGlnProThrLeuIleSerSerMetA la                             65707580                                                                      GlnSerGlnGlyGlnProLeuAlaSerGlnProProAlaValAspPro                              859095                                                                         TyrAspMetProGlyThrSerTyrSerThrProGlyLeuSerAlaTyr                             100105110                                                                     SerThrGlyGlyAlaSerGlySerGlyGlyProSerThrSerThrThr                               115120125                                                                    ThrSerGlyProValSerAlaArgProAlaArgAlaArgProArgArg                              130135140                                                                     ProArgGluGluThrLeuThrProGlu GluGluGluLysArgArgVal                             145150155160                                                                  ArgArgGluArgAsnLysLeuAlaAlaAlaLysCysArgAsnArgArg                              1651 70175                                                                    ArgGluLeuThrAspArgLeuGlnAlaGluThrAspGlnLeuGluGlu                              180185190                                                                     GluLysAlaGluLeuGluSerGluIleAlaGluLeuG lnLysGluLys                             195200205                                                                     GluArgLeuGluPheValLeuValAlaHisLysProGlyCysLysIle                              210215220                                                                     ProTyrGlu GluGlyProGlyProGlyProLeuAlaGluValArgAsp                             225230235240                                                                  LeuProGlySerThrSerAlaLysGluAspGlyPheGlyTrpLeuLeu                               245250255                                                                    ProProProProProProProLeuProPheGlnSerSerArgAspAla                              260265270                                                                     ProProAsnLeuThrAla SerLeuPheThrHisSerGluValGlnVal                             275280285                                                                     LeuGlyAspProPheProValValSerProSerTyrThrSerSerPhe                              290295 300                                                                    ValLeuThrCysProGluValSerAlaPheAlaGlyAlaGlnArgThr                              305310315320                                                                  SerGlySerGluGlnProSerAspProLeuAsnSerProS erLeuLeu                             325330335                                                                     AlaLeu                                                                    

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

We claim:
 1. An isolated polynucleotide encoding a trans-repressing FosB2 protein, wherein the protein is characterized by:(a) having a leucine zipper domain; and (b) forming a heterodimer with a Jun related protein, wherein the heterodimer is capable of:(i) binding to an AP-1 site; and (ii) suppressing transcriptional transactivation of a promoter containing the AP-1 site; and (c) having an amino acid sequence of Sequence ID No. 2, wherein amino acids 238-284 are deleted therefrom.
 2. The polynucleotide of claim 1, wherein the Jun related protein is selected from the group consisting of c-Jun, Jun B and Jun D.
 3. A polynucleotide of claim 1, wherein the polynucleotide is DNA.
 4. A host cell containing the polynucleotide of claim
 1. 5. A recombinant expression vector containing the polynucleotide of claim
 1. 6. The vector of claim 5, which is a colloidal dispersion system.
 7. The vector of claim 6, wherein the colloidal dispersion system is a liposome.
 8. The vector of claim 7, wherein the liposome is essentially target specific.
 9. The vector of claim 8, wherein the liposome is anatomically targeted.
 10. The vector of claim 8, wherein the liposome is mechanistically targeted.
 11. The vector of claim 10, wherein the mechanistic targeting is passive.
 12. The vector of claim 10, wherein the mechanistic targeting is active.
 13. The vector of claim 12, wherein the liposome is actively targeted by coupling with a moiety selected from the group consisting of a sugar, a glycolipid, and a protein.
 14. The vector of claim 13, wherein the protein moiety is an antibody.
 15. The vector of claim 5, which is a virus.
 16. The vector of claim 15, wherein the virus is an RNA virus.
 17. The vector of claim 11, wherein the RNA virus is a retrovirus.
 18. The vector of claim 17, wherein the retrovirus is essentially target specific.
 19. The vector of claim 18, wherein the moiety for target specificity is selected from the group consisting of a sugar, a glycolipid, and a protein.
 20. The vector of claim 19, wherein the protein is an antibody. 